Over the past half century scientists have learned much about how cells detect physical and chemical cues in their environment. Typically, cell signaling requires a plasma membrane receptor, an intracellular transducer and a downstream effector such as a protein kinase. Protein kinases are well known to phosphorylate downstream targets such as transcription factors, which drive new gene transcription. Most kinases also phosphorylate upstream components leading to positive or negative feedback. In this way, some signals become amplified while others become diminished. Familiar examples of negative feedback include desensitization to odors, light, and many pharmaceuticals. Whereas past work has focused on feedback inhibition leading to desensitization, our proposed work will focus on three additional and important consequences of feedback regulation: I: Signal coordination; for example to limit inappropriate activation of a competing kinase pathway. II: Signal tuning; for example to convert a graded input to a switch-like output, or vice versa. III: Signal tracking; for example to allow cell growth or migration towards a gradient stimulus. Our investigation will center on the mitogen activated protein kinases (MAPKs), which are activated in response to diverse (and often competing) stimuli including hormones, stresses and cytokines. Among the best- characterized MAPK pathways are those found in yeast Saccharomyces cerevisiae, where they contribute to cell mating and the osmotic stress response. Our approach will capitalize on recent breakthroughs, including newer fluorescent sensors capable of tracking biological responses, as well as new microfluidics devices capable of tracking pathway activity in single cells. Comprehensive identification of MAPK substrates, and establishing the consequences of those phosphorylation events, will inform new predictive computational models. Our investigations will require multiple rounds of data collection, model building, model testing, and model refinement, and would therefore benefit greatly from the flexibility and stability provided by the R35 grant mechanism.